Methods and Compositions for Amplified Electrochemiluminescence Detection Assays

ABSTRACT

Disclosed herein are formulations, substrates, and arrays. Also disclosed herein are methods for manufacturing and using the formulations, substrates, and arrays. Also disclosed are methods for identifying peptide sequences useful for diagnosis and treatment of disorders, and methods for using the peptide sequences for diagnosis and treatment of disorders, e.g., celiac disorder. In certain embodiments, substrates and arrays comprise a porous layer for synthesis and attachment of polymers or biomolecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/768,196, filed Aug. 14, 2015, allowed, which is the National Stage ofInternational Patent Application No. PCT/US2014/016737, filed Feb. 17,2014, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/765,584, filed Feb. 15, 2013, each of which are incorporatedherein in their entirety.

BACKGROUND

A typical microarray system generally comprises biomolecular probes,such as DNA, proteins, or peptides, formatted on a solid planar surfacelike glass, plastic, or silicon chip, plus the instruments needed tohandle samples (automated robotics), to read the reporter molecules(scanners) and analyze the data (bioinformatic tools). Microarraytechnology can facilitate monitoring of many probes per squarecentimeter. Advantages of using multiple probes include, but are notlimited to, speed, adaptability, comprehensiveness and the relativelycheaper cost of high volume manufacturing. The uses of such an arrayinclude, but are not limited to, diagnostic microbiology, including thedetection and identification of pathogens, investigation ofanti-microbial resistance, epidemiological strain typing, investigationof oncogenes, analysis of microbial infections using host genomicexpression, and polymorphism profiles.

Electrochemiluminescence or electrogenerated chemiluminescence (ECL) isa kind of luminescence produced during electrochemical reactions insolutions. In electrogenerated chemiluminescence, electrochemicallygenerated intermediates undergo a highly exergonic reaction to producean electronically excited state that then emits light upon relaxation toa lower-level state. This wavelength of the emitted photon of lightcorresponds to the energy gap between these two states. ECL excitationcan be caused by energetic electron transfer (redox) reactions ofelectrogenerated species. Such luminescence excitation is a form ofchemiluminescence where one/all reactants are produced electrochemicallyon the electrodes.

ECL is usually observed during application of potential (several volts)to electrodes of electrochemical cell that contains solution ofluminescent species (polycyclic aromatic hydrocarbons, metal complexes,Quantum Dots or Nanoparticles) in aprotic organic solvent (ECLcomposition). In organic solvents both oxidized and reduced forms ofluminescent species can be produced at different electrodessimultaneously or at a single one by sweeping its potential betweenoxidation and reduction. The excitation energy is obtained fromrecombination of oxidized and reduced species.

In aqueous medium which is mostly used for analytical applicationssimultaneous oxidation and reduction of luminescent species is difficultto achieve due to electrochemical splitting of water itself so the ECLreaction with the coreactants is used. In the later case luminescentspecies are oxidized at the electrode together with the coreactant whichgives a strong reducing agent after some chemical transformations (theoxidative reduction mechanism).

There is a need for a platform which can simultaneously detect multipleanalytes of varying concentrations, Typical ELISA based assays have 4orders of magnitude and hence is restricted in detecting multipleanalytes varying from ug/ml to fg/ml. Recent advances inelectrochemiluminescence have pushed the limits of detection to pg/mlwith a dynamic range up to 4-5 orders in log scale. However,simultaneous detection of multiple analytes in varying concentration of6 or more magnitudes has not been possible due to limitation on the tagsused for electrochemiluminescence.

SUMMARY

The invention encompasses, in several aspects formulations, substrates,and arrays. The invention also includes methods of detecting analytesusing the formulations, substrates, and arrays.

In an aspect, the invention comprises methods of detecting a targetbiomolecule comprising contacting sample comprising said targetbiomolecule with a capture ligand, said capture ligand being immobilizedat a defined location on a substrate and capable of specifically bindingsaid target biomolecule thereby immobilizing said target biomolecule atsaid defined location on said substrate; contacting said immobilizedtarget biomolecule with a detection ligand, said detection ligandcapable of specifically binding to said immobilized target biomoleculeand having peroxidase activity thereby forming an immobilized targetbiomolecule-detection ligand complex; contacting said complex with atagging solution comprising an AECL tag under conditions that promotecovalent binding of a plurality of AECL tags to said complex; washingsaid substrate to remove unbound AECL tag from said substrate;contacting said substrate with a detection solution that reacts withsaid bound AECL tag to generate luminescence when a voltage is appliedto said defined location on said substrate; applying said voltage tosaid defined location on said substrate; and measuring luminescence fromsaid defined location on said substrate thereby detecting said targetbiomolecule.

In certain embodiments, the defined location on the substrate is amicroarray feature or a plurality of microarray features. In certainembodiments, the features can have an edge dimension between 50 nm and 1um, or between 50 nm and 100 nm or between 50 nm and 75 nm. In certainembodiments, the capture ligands are covalently bound to the definedlocation via a COOH or an NH2 moiety provided on the substrate.

In certain embodiments, the capture ligand and detection ligand compriseantibodies, peptides, proteins, or antigen binding proteins.

In certain embodiments, the sample can comprise blood, serum, plasma,saliva, urine, feces or cerebrospinal fluid (CSF).

In certain embodiments, the sample is obtained from a human subject.

In certain embodiments, the AECL tag comprises a metal chelate, a rareearth metal chelate, a ruthenium chelate, or tris(bipyridine)ruthenium(II). The AECL tag can further comprise tyramidebound to the metal chelate. In certain embodiments the AECL tagcomprises

wherein M^(n+) is Ru²⁺.

In certain embodiments, the invention includes AECL tag compositions aswell as kits and solutions for binding said AECL tag compositions totarget biomolecules and detecting their binding via an emittedluminescent signal.

In other embodiments, the invention includes solid state microarrays andpillar assemblies for mounting the microarrays and performing AECLassays. In certain embodiments, the solid state microarrays comprisechemically-functionalized surfaces comprising COOH or NH2 functionalgroups that can be covalently bound to capture ligands.

In some embodiments, the solid state microarrays of the presentinvention have an electrical potential difference between at least onepair of working and counter electrodes that generateselectrochemiluminescence from bound AECL tags.

In some embodiments, the solid state microarrays comprise at least 2, 4,8, 16, 32, 64, 100, 200, 500, 1000, 2000, 5000, 10,000, 15,000, 20,000,30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 1,000,000,1,500,000, 2,000,000 or more working electrodes.

In some aspects, the invention includes an assay plate for mounting asolid state microarray. The assay plate includes a pillar that includesa top surface and a bottom surface. The top surface includes a mountingsurface to receive a solid state microarray of the invention as well asat least one working electrode and one counter electrode configured tocontact and to be in electrical communication with a corresponding atleast one working electrode and counter electrode on a bottom surface ofthe solid state microarray. The bottom surface of the pillar includescontacts for supplying power to the at least one pillar workingelectrode and pillar counter electrode. In some embodiments, the assayplate includes a plurality of pillars, such as 24, 96, 384 or 1586pillars. In some embodiments, the pillars comprise at least 2, 4, 8, 16,32, 64, 100, 200, 500, 1000, 2000, 5000, 10,000, 15,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 1,000,000, 1,500,000,2,000,000 or more working electrodes.

In some embodiments, the invention includes an assembly that includes anassay plate with a microarray mounted on the surface of the assay platepillar such that the corresponding pillar and microarray working andcounter electrodes are in electrical contact. In yet other embodiments,the assemblies of the invention further include an assay cap, thatprovides pillar walls mounted on struts that slidably engage grooves onthe pillar mounts. When the cap and the assay plate are engaged, thepillar walls provide barriers for a reservoir that can hold assay fluidin contact with the microarray.

In yet other embodiments, the AECL assays of the invention have improveddetection limits, such that concentrations of target biomolecules in asample can be detected at limits on the order of 100 fg/mL, 10 fg/mL, or1 fg/mL.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows the structure of an embodiment of an amplifiedelectrochemiluminescent (AECL) tag comprising a metal chelate esterattached to tyramide.

FIG. 2 shows the prior art process of tyramide signal amplificationcatalyzed by horseradish peroxidase (HRP) attached to an antibody. Thetyramide is bound to a fluorescent marker. The HRP localized to thesecondary antibody catalyzes the binding of tyramide to electron richmoieties (predominantly tyrosine residues) in a target.

FIG. 3 shows a prior art sandwich ELISA to detect a captured targetbiomolecule using an ECL-tagged secondary antibody and application ofvoltage in the presence of tripropyl amine (TPA) to produce a detectablelight signal that can be used to quantitate captured target biomolecule.

FIG. 4 illustrates a sandwich ELISA embodiment of the present inventionto detect a captured target biomolecule using a secondaryantibody—horseradish peroxidase (HRP) conjugate. Upon addition of anAECL tag in the presence of hydrogen peroxide, the HRP catalyzesattachment of the AECL tag's tyramide moiety to electron rich targets(predominantly tyrosine residues) that are proximate to the boundsecondary antibody, thus labeling the captured target biomoleculecomplex with multiple AECL tags. Application of voltage in the presenceof TPA produces an amplified light signal that can be used to quantitatecaptured target biomolecule.

FIG. 5 illustrates a cross-sectional view, top view, and bottom view ofthe electrode and well configuration of an array. In an embodiment, thearray is used with the electrochemiluminescence detection methodsdescribed herein.

FIG. 6 shows a top and bottom view for chips (including an embodiment ofelectrode and well configurations) comprising 1 feature group, 4 featuregroups, or 16 feature groups.

FIG. 7 shows top and bottom view for chips comprising 1 pillar group, 4pillar groups, or 16 pillar groups.

FIG. 8 shows a detailed diagram of an AECL pillar mount (top left) ontowhich four AECL microarray assay chips are mounted. An AECL assay cap(top right) is used to cover the AECL assay plate (bottom right) whichas shown, includes 9 separate pillar mounts. The assay cap includespillar walls mounted on struts that engage grooves on the sides of theassay plate pillars. In connection with the top surface of the pillarmount, the pillar walls form a reservoir that retains the AECL assaysolution (see side view, bottom left).

FIG. 9 shows a diagram of AECL detection of analytes on a single pillaron an assay plate.

FIGS. 10A and 10B show steps in the AECL chip manufacturing process.

FIGS. 11A and 11B respectively show steps in the AECL pillar mountmanufacturing process and a top view of an AECL pillar mount accordingto an embodiment of the invention.

FIG. 12 compares results of ECL and AECL biochip assays for TNF-alphaaccording to an embodiment of the invention. Y-axis is luminescence inarbitrary units, X-axis is amount of TNF-alpha/mL in assayed samplesolution. Thus 100 ng on X-axis corresponds to a TNF-alpha concentrationof 100 ng/mL in the assayed sample solution.

DETAILED DESCRIPTION

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

As used herein the term “wafer” refers to a slice of semiconductormaterial, such as silicon or a germanium crystal generally used in thefabrication of integrated circuits. Wafers can be in a variety of sizesfrom, e.g., 25.4 mm (1 inch) to 300 mm (11.8 inches) along one dimensionwith thickness from, e.g., 275 μm to 775 μm.

As used herein the terms “biomolecule,” “polypeptide,” “peptide,” or“protein” are used interchangeably to describe a chain or polymer ofamino acids that are linked together by bonds. Accordingly, the term“peptide” as used herein includes a dipeptide, tripeptide, oligopeptide,and polypeptide. The term “peptide” is not limited to any particularnumber of amino acids. In some aspects, a peptide contains about 2 toabout 50 amino acids, about 5 to about 40 amino acids, or about 5 toabout 20 amino acids. A molecule, such as a protein or polypeptide,including an enzyme, can be a “native” or “wild-type” molecule, meaningthat it occurs naturally in nature; or it may be a “mutant,” “variant,”“derivative,” or “modification,” meaning that it has been made, altered,derived, or is in some way different or changed from a native moleculeor from another molecule such as a mutant.

As used herein the term “microarray” refers to a substrate on whichdifferent probe molecules of proteins (e.g., antibodies, antibodyfragments, or other polypeptide sequences) or specific DNA bindingsequences have been affixed at separate locations in an ordered mannerthus forming a microscopic array. Specific probes are present in largecopy number (e.g., 10{circumflex over ( )}6) within an array unit calleda feature. An array can be characterized by the feature density (e.g., #features/cm{circumflex over ( )}2), the total number of features, thelength of a feature edge, a feature area, or the separation betweenfeatures (sometimes referred to as the array's “pitch”).

As used herein the term “microarray system” refers to a system usuallycomprised of biomolecular probes formatted on a solid planar surfacelike glass, plastic or silicon chip plus the instruments needed tohandle samples (automated robotics), to read the reporter molecules(scanners) and analyze the data (bioinformatic tools).

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the type of non-covalent interactions thatoccurs between an immunoglobulin molecule (or variant thereof such as anscFv) and an antigen for which the immunoglobulin is specific.

As used herein the term “biological sample” refers to a sample derivedfrom biological tissue or fluid that can be assayed for an analyte(s) ofinterest. Such samples include, but are not limited to, sputum, amnioticfluid, blood, blood cells (e.g., white cells), tissue or fine needlebiopsy samples, urine, peritoneal fluid, and pleural fluid, or cellstherefrom. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes. Although the sampleis typically taken from a human patient, the assays can be used todetect analyte(s) of interest in samples from any organism (e.g.,mammal, bacteria, virus, algae, or yeast) or mammal, such as dogs, cats,sheep, cattle, and pigs. The sample may be pretreated as necessary bydilution in an appropriate buffer solution or concentrated, if desired.

As used herein, the term “assay” refers to a type of biochemical testthat measures the presence or concentration of a substance of interestin solutions that can contain a complex mixture of substances.

The term “subject” as used herein may refer to a human or any otheranimal having a disorder for testing, diagnosis or treatment.

The term “antigen” as used herein refers to a molecule that triggers animmune response by the immune system of a subject, e.g., the productionof an antibody by the immune system and/or activation of the cellulararm of the immune system (e.g., activation of phagocytes, natural killercells, and antigen-specific cytotoxic T-lymphocytes, along with releaseof various cytokines in response to an antigen). Antigens can beexogenous, endogenous or auto antigens. Exogenous antigens are thosethat have entered the body from outside through inhalation, ingestion orinjection. Endogenous antigens are those that have been generated withinpreviously-normal cells as a result of normal cell metabolism, orbecause of viral or intracellular bacterial infection. Auto antigens arethose that are normal protein or protein complex present in the hostbody but can stimulate an immune response.

As used herein the term “epitope” or “immunoactive regions” refers todistinct molecular surface features of an antigen capable of being boundby component of the adaptive immune system, e.g., an antibody or T cellreceptor. Antigenic molecules can present several surface features thatcan act as points of interaction for specific antibodies. Any suchdistinct molecular feature can constitute an epitope. Therefore,antigens have the potential to be bound by several distinct antibodies,each of which is specific to a particular epitope.

As used herein the term “antibody” or “immunoglobulin molecule” refersto a molecule naturally secreted by a particular type of cells of theimmune system: B cells. There are five different, naturally occurringisotypes of antibodies, namely: IgA, IgM, IgG, IgD, and IgE.

As used herein the term “immune-related molecule” refers to a biologicalmolecule involved in the activation or regulation of an immune response.These include, for example, an antibody, T cell receptor, or MEW complex(e.g., human leukocyte antigen).

As used herein, the term “inflammatory response molecule” refers tomolecules that signal or mediate an inflammatory response, e.g.,cytokines such as interleukin and tumor necrosis factor. Inflammatoryresponse molecules include, for example, pro-inflammatory molecules.

As used herein, the term “autoimmune disorder” refers to any of a largegroup of diseases characterized by abnormal functioning of the immunesystem that causes a subject's immune system to damage the subject's owntissues. Celiac disorder, lupus erythematosis, and rheumatoid arthritisare examples of autoimmune disorders. Autoimmune disorders may beinduced by environmental factors.

The term “percent identity” or “percent sequence identity,” in thecontext of two or more nucleic acid or polypeptide sequences, refer totwo or more sequences or subsequences that have a specified percentageof nucleotides or amino acid residues that are the same, when comparedand aligned for maximum correspondence, as measured using one of thesequence comparison algorithms described below (e.g., BLASTP and BLASTNor other algorithms available to persons of skill) or by visualinspection. Depending on the application, the percent “identity” canexist over a region of the sequence being compared, e.g., over afunctional domain, or, alternatively, exist over the full length of thetwo sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Compositions

AECL Tags and Secondary Antibody.

Also disclosed herein are compositions for amplifiedelectrochemiluminescent detection of biomolecules of interest on anarray. In an embodiment, a compound is provided that covalently links anelectrochemiluminescence (ECL) moiety with a signal amplification moietyto generate an amplified electrochemiluminescence (AECL) tag. FIG. 1.The AECL tag generates detectable electromagnetic radiation (i.e.,light) upon exposure to voltage in the presence of tripropyl amine(TPA). In an embodiment, the ECL moiety is a metal chelate ester. In anembodiment, the metal is a rare earth metal. In an embodiment the rareearth metal is Ruthenium (Ru). In an embodiment, the signalamplification moiety is tyramide. The use of the tyramide as part of theAECL tag provides for a minimal background. The AECL tag is used with anenzyme-conjugated antigen binding protein (e.g., an HRP-conjugatedantibody) resulting in highly localized enzyme-mediated AECL tagdeposition to improve detection of bound target molecules. See FIG. 4and accompanying description below.

Prior art tyramide signal amplification assays result in covalentbinding of labeled tyramide to tyrosine residues (e.g., on the secondaryantibody, target biomolecule and primary antibody) in the presence ofhorseradish peroxidase (HRP) and hydrogen peroxide. The label can be afluorescent tag, or a detectable reaction product e.g., an insolubleproduct produced by action of another enzyme such as alkalinephosphatase on a chromogenic substrate. See FIG. 2.

Prior art ECL assays use an ECL tag comprising a metal chelate estercovalently bound to a detection ligand such as, e.g., a secondaryantibody used in a sandwich ELISA format. The ECL tag emits light in thepresence of tripropyl amine (TPA) when exposed to an electric field(e.g., by supplying a voltage difference across a working electrode inelectrical communication with a binding complex comprising the capturedtarget biomolecule and a counter electrode), a phenomena calledelectrochemiluminescence (FIG. 3).

In some embodiments of the present invention, an antibody array isexposed to a sample comprising a biomolecule of interest. At least oneprimary antibody bound to the array surface binds to the biomolecule ofinterest. After washing the array, the array is exposed to a solution ofsecondary antibody conjugated to horseradish peroxidase (HRP), whereinthe secondary antibody binds to the biomolecule of interest. Afterwashing, the array is exposed to a solution comprising hydrogen peroxideand an AECL tag. AECL tags bind to complexes attached to the array whichcomprise primary antibodies bound to protein and secondary antibodyconjugated to HRP. The AECL tags comprise tyramide which binds totyrosine in the presence of HRP (conjugated to the secondary antibody).The array is then washed and exposed to tripropylamine (TPA), whichreacts with the metal chelate of the AECL tag to activate it, thuscausing it to generate chemiluminescence when exposed to a voltagepotential (e.g., a voltage potential from the array). Thus, underapplied voltage, the metal chelate ester generates anelectrochemiluminescent (ECL) output (FIG. 4). This method of AECLtagging improves the detection sensitivity by at least 10 fold to 1000fold as compared to commercially available ECL.

Substrates

Also disclosed herein are substrates. In some aspects a substratesurface is planar (i.e., 2-dimensional). In some aspects a substratesurface is functionalized with free carboxylic acid groups. In someaspects, a substrate surface is functionalized with free amine groups. Asurface that is functionalized with free amine groups may be convertedto free carboxylic acid groups by reacting with activating thecarboxylic acid groups of a molecule comprising at least two freecarboxylic acid groups (e.g., converting the carboxylic acid group to acarbonyl group using carbodiimide) and reacting the molecule with thefree amine groups attached to the surface of the substrate. In someembodiments, the molecule comprising multiple carboxylic acid groups issuccinic anhydride, polyethylene glycol diacid,benzene-1,3,5-tricarboxylic acid, benzenehexacarboxylic acid, orcarboxymethyl dextran.

In some aspects, a substrate can include a porous layer (i.e., a3-dimensional layer) comprising functional groups for binding a firstmonomer building block. In some aspects, a substrate surface comprisespillars for peptide attachment or synthesis. In some embodiments, aporous layer is added to the top of the pillars.

Porous Layer Substrates

Porous layers which can be used are flat, permeable, polymeric materialsof porous structure which have a carboxylic acid functional group (whichis native to the constituent polymer or which is introduced to theporous layer) for attachment of the first peptide building block. Forexample, a porous layer can be comprised of porous silicon withfunctional groups for attachment of a polymer building block attached tothe surface of the porous silicon. In another example, a porous layermay comprise a cross-linked polymeric material. In some embodiments, theporous layer may employ polystyrenes, saccharose, dextrans,polyacryloylmorpholine, polyacrylates, polymethylacrylates,polyacrylamides, polyacrylolpyrrolidone, polyvinylacetates,polyethyleneglycol, agaroses, sepharose, other conventionalchromatography type materials and derivatives and mixtures thereof. Insome embodiments, the porous layer building material is selected from:poly(vinyl alcohol), dextran, sodium alginate, poly(aspartic acid),poly(ethylene glycol), poly(ethylene oxide), poly(vinyl pyrrolidone),poly(acrylic acid), poly(acrylic acid)-sodium salt, poly(acrylamide),poly(N-isopropyl acrylamide), poly(hydroxyethyl acrylate), poly(acrylicacid), poly(sodium styrene sulfonate),poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, andcellulose derivatives. Preferably the porous layer has a porosity of10-80%. In an embodiment, the thickness of the porous layer ranges from0.01 μm to about 1,000 μm. Pore sizes included in the porous layer mayrange from 2 nm to about 100 μm.

According to another aspect of the present invention there is provided asubstrate comprising a porous polymeric material having a porosity from10-80%, wherein reactive groups are chemically bound to the poresurfaces and are adapted in use to interact, e.g. by binding chemically,with a reactive species, e.g., deprotected monomeric building blocks orpolymeric chains. In an embodiment the reactive group is a carboxylicacid group. The carboxylic acid group is free to bind, for example, anunprotected amine group of a peptide or polypeptide.

In an embodiment, the porous layer is in contact with a support layer.The support layer comprises, for example, metal, plastic, silicon,silicon oxide, or silicon nitride. In another embodiment, the porouslayer may be in contact with a patterned surface, such as on top ofpillar substrates described below.

AECL Chip Substrates

Semiconductor manufacturing processes can be used to generate AECL chipsthat have solid state electrode circuitry built into one surface of asilicon substrate and biomolecular features present, usually patternedas an array on the opposite face of the substrate on the workingelectrode surface. Any technique useful for patterning biomolecularfeatures such as peptides or proteins can be used, including those forsynthesizing peptides in situ in an N→C or C→N configuration, or fortethering whole proteins at defined locations using carbodiimide basedchemistries such as those described in co-owned cases WO2013/119845 andPCT/US2013/070207, and described below.

The AECL chip manufacturing process results in production of anintegrated biochip sensor device that is attached to a controller usedto drive voltage feeds to reference and working electrodes in order toexcite a chemiluminescent signal, which, according to embodiments of thepresent invention, is amplified.

The controller can also be programmed and used to drive imageacquisition and data storage for the assay results. Additional detailsrelating to AECL chip substrate manufacture and the use of the resultingchips in AECL assays is described in greater detail in Examples 1-6,below.

Arrays

Also disclosed herein are arrays. In some aspects, the surface of thearray is functionalized with free carboxylic acids. In some aspects, thefree carboxylic acids are activated to bind to amine groups, e.g.,during polypeptide synthesis on the surface of the array. In someembodiments, the surface density of free carboxylic acid groups on thearray is greater than 10/cm², 100/cm², 1,000/cm², 10,000/cm²,100,000/cm², 1,000,000/cm², or 10,000,000/cm².

In some aspects, an array can be a three-dimensional array, e.g., aporous array comprising features attached to the surface of the porousarray. In some aspects, the surface of a porous array includes externalsurfaces and surfaces defining pore volume within the porous array. Insome aspects, a three-dimensional array can include features attached toa surface at positionally-defined locations, said features eachcomprising: a collection of peptide chains of determinable sequence andintended length. In an embodiment, within an individual feature, thefraction of peptide chains within said collection having the intendedlength is characterized by an average coupling efficiency for eachcoupling step of greater than 98%.

In some aspects, the average coupling efficiency for each coupling stepis at least 98.5%. In some aspects, the average coupling efficiency foreach coupling step is at least 99%. In some aspects, the averagecoupling efficiency for each coupling step is at least 90, 91, 92, 93,94, 95, 96, 97, 98, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100%.

In some aspects, each peptide chain is from 5 to 60 amino acids inlength. In some aspects, each peptide chain is at least 5 amino acids inlength. In some aspects, each peptide chain is at least 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. In someaspects, each peptide chain is less than 5, at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or greater than60 amino acids in length. In some aspects, each peptide chain comprisesone or more L amino acids. In some aspects, each peptide chain comprisesone or more D amino acids. In some aspects, each peptide chain comprisesone or more naturally occurring amino acids. In some aspects, eachpeptide chain comprises one or more synthetic amino acids.

In some aspects, an array can include at least 1,000 different peptidechains attached to the surface. In some aspects, an array can include atleast 10,000 different peptide chains attached to the surface. In someaspects, an array can include at least 100, 500, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10,000, or greater than 10,000 differentpeptide chains attached to the surface (or any integer in between).

In some aspects, each of the positionally-defined locations is at adifferent, known location that is physically separated from each of theother positionally-defined locations. In some aspects, each of thepositionally-defined locations is a positionally-distinguishablelocation. In some aspects, each determinable sequence is a knownsequence. In some aspects, each determinable sequence is a distinctsequence.

In some aspects, the features are covalently attached to the surface. Insome aspects, said peptide chains are attached to the surface through alinker molecule or a coupling molecule.

In some aspects, the features comprise a plurality of distinct, nested,overlapping peptide chains comprising subsequences derived from a sourceprotein having a known sequence. In some aspects, each peptide chain inthe plurality is substantially the same length. In some aspects, eachpeptide chain in the plurality is the same length. In some aspects, eachpeptide chain in the plurality is at least 5 amino acids in length. Insome aspects, each peptide chain in the plurality is at least 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. In someaspects, each peptide chain in the plurality is less than 5, at least 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,or greater than 60 amino acids in length. In some aspects, at least onepeptide chain in the plurality is at least 5 amino acids in length. Insome aspects, at least one peptide chain in the plurality is at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. Insome aspects, at least one peptide chain in the plurality is less than5, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, or greater than 60 amino acids in length. In someaspects, each polypeptide in a feature is substantially the same length.In some aspects, each polypeptide in a feature is the same length. Insome aspects, the features comprise a plurality of peptide chains eachhaving a random, determinable sequence of amino acids.

Carboxylic Acid Activation Solutions

Disclosed herein are activation formulations for activating carboxylicacid so that it reacts with a free amino group of a biomolecule, e.g., apeptide. An activation formulation can include components such as acarboxylic acid group activating compound and a solvent. In anembodiment, the carboxylic acid group activating compound is acarbodiimide or a carbodiimide precursor. In some aspects, thecarbodiimide is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In someembodiments, the carboxylic acid group activating compound isN-Hydroxysuccinimide (NHS). In some embodiments, the carboxylic acidgroup activating compound is selected from:1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC],N-hydroxysuccinimide [NHS], 1,3-Diisopropylcarbodiimide [DIC],hydroxybenzotriazole (HOBt),(O-(7-azabenzotriazol-1-yl)-N,N,N,N′-tetramethyluroniumhexafluorophosphate) [HATU],benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate[PyBOP], and N,N-Diisopropylethylamine [DIEA]. In some embodiments, thesolvent is water. In some embodiments, the solvent isN-methylpyrrolidone (NMP). In some embodiments, the carboxylic acidgroup activating compound converts the carboxylic acid to a carbonylgroup (i.e., carboxylic acid group activation). In some embodiments, thecarboxylic acid group is activated for 5, 10, 15, 20, 30, 45, or 60minutes after exposure to an activation formulation.

In some aspects, the activation formulation comprises 4% by weight of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 2% by weight ofN-hydroxysuccinimide (NHS) dissolved in deionized water. In someaspects, the activation formulation comprises 4% by weight of1,3-Diisopropylcarbodiimide (DIC) and 2% by weight ofhydroxybenzotriazole (HOBt) dissolved in NMP. In some aspects, theactivation formulation comprises 4% by weight of(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) (HATU) and 2% by weight ofN,N-Diisopropylethylamine (DIEA) dissolved in NMP. In some aspects, theactivation formulation comprises 4% by weight ofBenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP) and 2% by weight of N,N-Diisopropylethylamine (DIEA) dissolvedin NMP.

In some embodiments, the carboxylic acid group activating compound is acarbodiimide precursor. In one aspect, the carbodiimide precursor isconverted to a carbodiimide through exposure to radiation, e.g.,ultraviolet radiation. In an embodiment, the carbodiimide precursor is athione. The carbodiimide precursor may also be referred to as aphotoactivated carbodiimide. In an embodiment, photoactivatedcarbodiimides are used to provide site-specific activation of carboxylicacid groups on an array by spatially controlling exposure of thephotoactivated carbodiimide solution to electromagnetic radiation at apreferred activation wavelength. In some embodiments, the preferredactivation wavelength is 248 nm.

In an embodiment, the carbodiimide precursor is a thione that isconverted to carbodiimide via photoactivation. In one aspect, the thioneis converted to a hydroxymethyl phenyl carbodiimide after exposure toelectromagnetic radiation. In some embodiments, the thione is4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione,1-ethyl-4-dimethylaminopropyl tetrazole 5-thione,1,3-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-5-thione,4-cyclohexyl-1H-tetrazole-5(4H)-thione, or 1-phenyl-4-(piperidinomethyl)tetrazole-5(4H)-thione.

In some embodiments, the activation solution comprises a carbodiimideprecursor, a solvent, and a polymer. In an embodiment, the carbodiimideprecursor is4,5-dihydro-4-(hydroxymethyl)-1-phenyl-1H-tetrazole-5-thione,1-ethyl-4-dimethylaminopropyl tetrazole 5-thione, or1,3-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)-5-thione. In some aspects,the carbodiimide precursor is present in the activation solution at aconcentration of 2.5% by weight. In some aspects the carbodiimideprecursor is present in the activation solution at a concentration of0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3., 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or 5.0% by weight of the totalformulation concentration.

In some embodiments, the solvent is water. In some aspects, the solventis about 80-90% by weight of the total formulation concentration. Insome aspects, the solvent is about less than 70, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or greater than 99% by weight of the totalformulation concentration.

In some aspects, a polymer is a polyvinyl pyrrolidone and/or a polyvinylalcohol. In some aspects, a polymer is about 0.5-5% by weight of thetotal formulation concentration. In some aspects, a polymer is aboutless than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3., 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than5.0% by weight of the total formulation concentration.

In some aspects, a coupling reagent is a carbodiimide. In some aspects,a coupling reagent is a triazole. In some aspects, a coupling reagent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. In some aspects, acoupling reagent is about 0.5-5% by weight of the total formulationconcentration. In some aspects, a coupling reagent is about less than0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3.,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% byweight of the total formulation concentration.

Linker Formulations

Also disclosed herein is a linker formulation. A linker formulation caninclude components such as a solvent, a polymer, a linker molecule, anda coupling reagent. In some aspects, the polymer is 1% by weightpolyvinyl alcohol and 2.5% by weight poly vinyl pyrrolidone, the linkermolecule is 1.25% by weight polyethylene oxide, the coupling reagent is1% by weight 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and thesolvent includes water. In some aspects, the polymer is 0.5-5% by weightpolyvinyl alcohol and 0.5-5% by weight poly vinyl pyrrolidone, thelinker molecule is 0.5-5% by weight polyethylene oxide, the couplingreagent is 0.5-5% by weight 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent includes water.

In some aspects, the solvent is water, an organic solvent, or acombination thereof. In some aspects, the organic solvent is N Methylpyrrolidone, Di methyl formamide, Di chloromethane, Di methyl sulfoxide,or a combination thereof. In some aspects, the solvent is about 80-90%by weight of the total formulation concentration. In some aspects, thesolvent is about less than 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or greater than 99% by weight of the total formulationconcentration.

In some aspects, a polymer is a polyvinyl pyrrolidone and/or a polyvinylalcohol. The general structure of polyvinyl alcohol is as follows, wheren is any positive integer greater than 1:

In some aspects, a polymer is about 0.5-5% by weight of the totalformulation concentration. In some aspects, a polymer is about less than0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3.,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% byweight of the total formulation concentration.

A linker molecule can be a molecule inserted between a surface disclosedherein and peptide that is being synthesized via a coupling molecule. Alinker molecule does not necessarily convey functionality to theresulting peptide, such as molecular recognition functionality, but caninstead elongate the distance between the surface and the peptide toenhance the exposure of the peptide's functionality region(s) on thesurface. In some aspects, a linker can be about 4 to about 40 atoms longto provide exposure. The linker molecules can be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units(PEGs), diamines, diacids, amino acids, and combinations thereof.Examples of diamines include ethylene diamine and diamino propane.Alternatively, linkers can be the same molecule type as that beingsynthesized (e.g., nascent polymers or various coupling molecules), suchas polypeptides and polymers of amino acid derivatives such as forexample, amino hexanoic acids. In some aspects, a linker molecule is amolecule having a carboxylic group at a first end of the molecule and aprotecting group at a second end of the molecule. In some aspects, theprotecting group is a t-Boc protecting group or an Fmoc protectinggroup. In some aspects, a linker molecule is or includes an arylacetylene, a polyethyleneglycol, a nascent polypeptide, a diamine, adiacid, a peptide, or combinations thereof. In some aspects, a linkermolecule is about 0.5-5% by weight of the total formulationconcentration. In some aspects, a linker molecule is about less than0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3.,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or greater than 5.0% byweight of the total formulation concentration.

The unbound portion of a linker molecule, or free end of the linkermolecule, can have a reactive functional group which is blocked,protected, or otherwise made unavailable for reaction by a removableprotective group, e.g., t-Boc or F-Moc as noted above. The protectinggroup can be bound to a monomer, a polymer, or a linker molecule toprotect a reactive functionality on the monomer, polymer, or linkermolecule. Protective groups that can be used include all acid and baselabile protecting groups. For example, peptide amine groups can beprotected by t-butoxycarbonyl (t-BOC or BOC) or benzyloxycarbonyl (CBZ),both of which are acid labile, or by 9-fluorenylmethoxycarbonyl (FMOC),which is base labile.

Additional protecting groups that can be used include acid labile groupsfor protecting amino moieties: tert-amyloxycarbonyl,adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl,2-(p-biphenyl)propyl(2)oxycarbonyl,2-(p-phenylazophenylyl)propyl(2)oxycarbonyl,alpha,alpha-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl,2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl,furfuryloxycarbonyl, triphenylmethyl (trityl),p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl,diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl,and 1-naphthylidene; as base labile groups for protecting aminomoieties: 9 fluorenylmethyloxycarbonyl, methyl sulfonylethyloxycarbonyl,and 5-benzisoazolylmethyleneoxycarbonyl; as groups for protecting aminomoieties that are labile when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; as groups for protecting aminomoieties that are labile when oxidized: (ethylthio)carbonyl; as groupsfor protecting amino moieties that are labile to miscellaneous reagents,the appropriate agent is listed in parenthesis after the group:phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl(2-aminothiophenol); acid labile groups for protecting carboxylic acids:tert-butyl ester; acid labile groups for protecting hydroxyl groups:dimethyltrityl. (See also, Greene, T. W., Protective Groups in OrganicSynthesis, Wiley-Interscience, NY, (1981)).

Arrays with electrodes for applying voltage to the surface of the array

Also described herein is a system for enhancing electrochemiluminescenceon a silicon platform to provide a significant increase in the number ofworking electrodes/counter electrodes that can be accommodated for asingle assay pillar. FIG. 5 shows one embodiment of a configuration ofworking electrodes and counter electrode across two wells on an array orpillar. In cross-sectional view (FIG. 5 top panel), it is a four layerintegrated circuit. The top layer defines location of working electrodeand counter electrode, which are isolated by dielectric material.Peptides or other capture ligands (e.g., antigen binding proteins suchas antibodies, scFvs or the like) are synthesized in situ or otherwisecoupled (e.g., using carbodiimide chemistry) on the surface of a workingelectrode. The middle two layers are metal interconnection layers toconnect and group counter electrode or working electrode, which are alsoisolated by dielectric material. The bottom layer includes the outputsof working electrode and counter electrode, which are connected to apower supply or control unit. In top view (FIG. 5, middle panel), eacharray feature has its own working electrode and counter electrode, whichare used to generate an electrical potential difference when theelectrodes are powered. Bottom view (FIG. 5, bottom panel) shows anexample of electrode output. According to design choices for featuregrouping, the electrode output layout will differ, as described below.

FIG. 6 shows a view of 16 features on a microarray chip according to 3different embodiments. In an embodiment, 1 “feature group” is detected,allowing detection and quantitation of up to one biomolecule of interest(top left). In another embodiment 4 “feature groups” are detected,allowing detection and quantitation of up to four biomolecules ofinterest (top middle). In another embodiment 16 “feature groups” aredetected, allowing detection and quantitation of up to 16 biomoleculesof interest (top right). Working and counter electrode outputs are shownin the chip bottom view. In some embodiments, the microarray chipcomprises 2, 4, 8, 16, 32, 64, 100, 200, 500, 1000, 2000, 5000, 10,000,15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000,1,000,000, 1,500,000, 2,000,000 or more working electrodes or electrodepairs. In some embodiments, the microarray chip includes multipleelectrode pairs wherein each feature associated with these multipleelectrode pairs includes the same capture ligand to capture the sameanalyte. In some embodiments the microarray includes the same number ofcounter electrodes as working electrode and is configured to detect asingle analyte or multiple distinct analytes on a chip. By having thesame number of counter and working electrodes, the voltage applied to afeature can be precisely controlled. In some embodiments, 500 or moreanalytes (e.g., for triplicate measurements, one third the total ofnumber of features, such as described above) that can be detected on onechip. Statistically robust data can be obtained by having multiplefeatures having the same capture ligand.

Microarrays of the present invention can include features as small as 50nm on edge because the amplified ECL tag system produces extremely highsignal to noise ratios. In some embodiments, the features have an edgedimension between 50 nm and 1 um. In other embodiments, the featureshave an edge dimension between 50 nm and 100 nm. In yet otherembodiments, the edge dimension is between 50 nm and 75 nm. We havedemonstrated reliable and accurate detection of target biomolecules toAECL microarrays having features as small as 50 nm on edgenotwithstanding the reduced number of capture ligands and bound targetsas compared to larger features. Assuming constant capture liganddensity, the number of capture ligands per feature is a function offeature area. Thus as compared to a square feature having an edge lengthof 100 nm, a 50 nm feature would have ¼ the number of capture ligands.By using the AECL approach and a 50 nm feature length, a 3 mm×3 mmmicroarray chip can typically include anywhere from 200,000 to 2,000,000features.

In FIG. 7, an AECL assay plate array is shown in an embodimentcomprising a set of 16 pillars, each of which receives and suppliesvoltage to four separate AECL microarray chips. Three different pillargroupings are shown, in which the AECL assay plate is divided into 1pillar group (left panel), 4 pillar groups (middle panel), or 16 pillargroups (right panel). The bottom view shows, according to eachembodiment, working and counterelectrode configurations for each numberof pillar groups.

FIG. 8 shows a detailed view of an AECL assay plate, according to anembodiment of the invention. The counter electrodes (“count electrode”in FIG. 8) and working electrodes are attached to the array at the topof the pillar mount (top left panel). The array can be divided intomultiple sections, with a counter electrode and working electrodeattached to each. An AECL assay cap (top right panel) mounts onto anexemplary AECL assay plate (bottom right panel), that includes nine AECLpillar mounts (i.e., the structure shown in the top left panel), each ofwhich, in this example, receives and supplies voltage to four separateAECL microarray chips. The assay cap includes pillar walls that containthe assay solution when the cap is mounted onto the assay plate. SeeFIG. 8 bottom left (side view) and top right (assay cap). Bottom leftpanel (side view) shows AECL microarray (“chip”) mounted on pillar andcovered with AECL assay solution. The number of assay pillars includedin an assay plate can be selected according to the number of differentassays sought to be carried out. For example, the assay plate caninclude 24, 96, 384 or 1586 pillars in conformity with standardmicrotiter plate configurations.

FIG. 9 diagrams an exemplary system for detecting biomolecules bound toan AECL microarray chip, according to an embodiment of the invention.Voltage is applied to selected working and counter electrode leads arrayvia a main controller attached to a power supplier controller. Afterapplying voltage to the array, AECL tags illuminate features comprisingbound target biomolecules of interest, as described in thisspecification. The luminescence is. The chip is placed on a scannerstage and luminescence is optically detected.

In an embodiment, the system optics employ a wet (i.e., immersion)microscope lens stepping and scanning at a very minimal distance fromthe pillar top (approx. 0.5 mm) to increase the numerical aperture andreduces the loss of light from AECL. The control of the system can becompletely automated such that individual electrodes can be turned onand off, at times precisely coinciding with optimal placement of arrayfeatures with respect to the optics for image capture thus minimizingloss of signal from signal decay of light.

Methods of Manufacturing Arrays

Methods of Attaching Biomolecules to an Array

Also disclosed herein are methods for manufacturing arrays. In someaspects, capture ligands positioned at predetermined locations onmicroarrays disclosed herein can be synthesized in situ on a surface,e.g., a substrate disclosed herein. In some instances, the arrays aremade using photolithography. For example, the substrate is contactedwith a photoactive coupling solution. Masks can be used to controlradiation or light exposure to specific locations on a surface providedwith free linker molecules or free coupling molecules having protectinggroups. In the exposed locations, the protecting groups are removed,resulting in one or more newly exposed reactive moieties on the couplingmolecule or linker molecule. The desired linker or coupling molecule isthen coupled to the unprotected attached molecules, e.g., at thecarboxylic acid group. The process can be repeated to synthesize a largenumber of features in specific or positionally-defined locations on asurface (see, for example, U.S. Pat. No. 5,143,854 to Pirrung et al.,U.S. Patent Application Publication Nos. 2007/0154946 (filed on Dec. 29,2005), 2007/0122841 (filed on Nov. 30, 2005), 2007/0122842 (filed onMar. 30, 2006), 2008/0108149 (filed on Oct. 23, 2006), and 2010/0093554(filed on Jun. 2, 2008), each of which is herein incorporated byreference).

In some aspects, a method of producing a three-dimensional (e.g.,porous) array of features, can include obtaining a porous layer attachedto a surface; and attaching the features to the porous layer, saidfeatures each comprising a collection of peptide chains of determinablesequence and intended length, wherein within an individual feature, thefraction of peptide chains within said collection having the intendedlength is characterized by an average coupling efficiency for eachcoupling step of at least about 98%. In some aspects, the features areattached to the surface using a photoactive coupling formulation,comprising a photoactive compound, a coupling molecule, a couplingreagent, a polymer, and a solvent. In some aspects, the features areattached to the surface using a photoactive coupling formulationdisclosed herein. In some aspects, the photoactive coupling formulationis stripped away using water.

In an embodiment, described herein is a process of manufacturing anarray. A surface comprising attached carboxylic acid groups is provided.The surface is contacted with a photoactive coupling solution comprisinga photoactive compound, a coupling molecule, a coupling reagent, apolymer, and a solvent. The surface is exposed to ultraviolet light in adeep ultra violet scanner tool according to a pattern defined by aphotomask, wherein the locations exposed to ultraviolet light undergophoto base generation due to the presence of a photobase generator inthe photoactive coupling solution. The expose energy can be from 1mJ/cm² to 100 mJ/cm² in order to produce enough photobase.

The surface is post baked upon exposure in a post exposure bake module.Post exposure bake acts as a chemical amplification step. The bakingstep amplifies the initially generated photobase and also enhances therate of diffusion to the substrate. The post bake temperature can varybetween 75° C. to 115° C., depending on the thickness of the poroussurface, for at least 60 seconds and not usually exceeding 120 seconds.The free carboxylic acid group is coupled to the deprotected amine groupof a free peptide or polypeptide, resulting in coupling of the freepeptide or polypeptide to the carboxylic acid group attached to thesurface. This surface can be a porous surface. The synthesis of peptidescoupled to a carboxylic acid group attached to the surface occurs in anN→C synthesis orientation, with the amine group of free peptidesattaching to carboxylic acid groups bound to the surface of thesubstrate. Alternatively, a diamine linker may be attached to a freecarboxylic acid group to orient synthesis in a C→N direction, with thecarboxylic acid group of free peptides attaching to amine groups boundto the surface of the substrate.

The photoactive coupling solution can now be stripped away. In someaspects, provided herein is a method of stripping the photoresistcompletely with DI water. This process is accomplished in a developermodule. The wafer is spun on a vacuum chuck for, e.g., 60 seconds to 90seconds and deionized water is dispensed through a nozzle for about 30seconds.

The photoactive coupling formulation may be applied to the surface in acoupling spin module. A coupling spin module can typically have 20nozzles or more to feed the photoactive coupling formulation. Thesenozzles can be made to dispense the photoactive coupling formulation bymeans of pressurizing the cylinders that hold these solutions or by apump that dispenses the required amount. In some aspects, the pump isemployed to dispense 5-8 cc of the photoactive coupling formulation ontothe substrate. The substrate is spun on a vacuum chuck for 15-30 secondsand the photoactive coupling formulation is dispensed. The spin speedcan be set to 2000 to 2500 rpm.

Optionally, a cap film solution coat is applied on the surface toprevent the unreacted amino groups on the substrate from reacting withthe next coupling molecule. The cap film coat solution can be preparedas follows: a solvent, a polymer, and a coupling molecule. The solventthat can be used can be an organic solvent like N methyl pyrrolidone, dimethyl formamide, or combinations thereof. The capping molecule istypically acetic anhydride and the polymer can be Poly vinylpyrrolidone, polyvinyl alcohol, polymethyl methacrylate, poly (methyliso propenyl) ketone, or poly (2 methyl pentene 1 sulfone). In someembodiments, the capping molecule is ethanolamine

This process is done in a capping spin module. A capping spin module caninclude one nozzle that can be made to dispense the cap film coatsolution onto the substrate. This solution can be dispensed throughpressurizing the cylinder that stores the cap film coat solution orthrough a pump that precisely dispenses the required amount. In someaspects, a pump is used to dispense around 5-8 cc of the cap coatsolution onto the substrate. The substrate is spun on a vacuum chuck for15-30 s and the coupling formulation is dispensed. The spin speed can beset to 2000 to 2500 rpm.

The substrates with the capping solution are baked in a cap bake module.A capping bake module is a hot plate set up specifically to receivewafers just after the capping film coat is applied. In some aspects,provided herein is a method of baking the spin coated capping coatsolution in a hot plate to accelerate the capping reactionsignificantly. Hot plate baking generally reduces the capping time foramino acids to less than two minutes.

The byproducts of the capping reaction are stripped in a strippermodule. A stripper module can include several nozzles, typically up to10, set up to dispense organic solvents such as acetone, iso propylalcohol, N methyl pyrrolidone, Di methyl formamide, DI water, etc. Insome aspects, the nozzles can be designated for acetone followed by isopropyl alcohol to be dispensed onto the spinning wafer. The spin speedis set to be 2000 to 2500 rpm for around 20 s.

This entire cycle can be repeated as desired with different couplingmolecules each time to obtain a desired sequence.

In some aspects, an array comprising a surface of free carboxylic acidsis used to synthesize polypeptides in an N->C orientation. In anembodiment, the carboxylic acids on the surface of the substrate areactivated (e.g., converted to a carbonyl) to allow them to bind to freeamine groups on an amino acid. In an embodiment, activation ofcarboxylic acids on the group of the surface can be done by addition ofa solution comprising a carbodiimide or succinimide to the surface ofthe array. In some embodiments, carboxylic acids can be activated byaddition of a solution comprising1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC],N-hydroxysuccinimide [NHS], 1,3-Diisopropylcarbodiimide [DIC],hydroxybenzotriazole (HOBt),(O-(7-azabenzotriazol-1-yl)-N,N,N,N′-tetramethyluroniumhexafluorophosphate) [HATU],benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate[PyBOP], or N,N-Diisopropylethylamine [DIEA] to the surface of thearray. The activation solution is washed away and the surface of thearray is prepared for addition of an amino acid layer (i.e., one aminoacid at each activated carboxylic acid group). Carboxylic acid groupsremain activated for up to 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.

Addition of a solution comprising an amino acid with a free amine groupto the activated carboxylic acid surface of the array results in bindingof a single amino acid to each carboxylic acid group. In someembodiments, the amino acid comprises an amino acid with protected aminegroups. Using a photosensitive chemical reaction, the protecting groupcan be removed from the amine group of selected amino acids atsite-specific locations using a reticle. For example, Fmoc-protectedamino acids are mixed in a solution comprising a photobase. Uponexposure of the solution on the array to a specific frequency of lightat site-specific locations, the photobase will release a base which willdeprotect the amino acid, resulting in coupling of the amino acid to theactivated carboxylic acid group on the surface of the array. Anothermethod of generating a base is through the use of a photoacid generator.In some embodiments, the photoacid generator is N-Boc-piperidine or1-Boc-4-piperazine.

After a completed layer of amino acids is coupled, remaining uncoupledactivated carboxylic acids are capped to prevent nonspecific binding ofamino acids on subsequent synthesis steps. The steps of activation,addition of an amino acid layer, and capping are repeated as necessaryto synthesize the desired polypeptides at specific locations on thearray.

In an embodiment, peptides synthesized in the N->C terminus directioncan be capped with a diamine molecule to enhance binding properties ofselected polypeptide sequences to a biological molecule, e.g., anantibody. In other aspects, peptides synthesized in the C->N directioncan be capped with a dicarboxylic acid molecule to enhance bindingproperties of selected sequences to a biological molecule.

While synthesizing polypeptides in parallel on the surface of an array,the method described herein ensures complete activation of carboxylicacid on the surface of the array. Due to stability of the activatedester for an extended period of time, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more couplingcycles may be completed after a single activation step (e.g., to couplean entire layer of 2-25 or more different amino acids at differentlocations on the array). As the coupling occurs during hard bake and dueto the presence of excess amino acid in the solution, complete 100%deprotection of Fmoc-protected amino acid may not be required forsignificantly high coupling yields. After addition of all amino acidsand capping, all free activated carboxylic acids are either coupled orcapped, thus resulting in high efficiency and accuracy of polypeptidesynthesis.

In an embodiment, proteins, polypeptides, or other molecules areattached to the activated carboxylic acid group on the surface of thearray. After activation of carboxylic acid groups on the array, asolution comprising proteins, polypeptides, or other molecules with afree amine group are added to the surface of the array. The amine groupbinds to the activated carboxylic acid group, thus attaching theprotein, polypeptide, or other molecule to the array. In an embodiment,this method is used to attach antibodies to the surface of the array. Inon embodiment, the amine groups are protected, and subsequentlydeprotected on the surface of the chip. In an embodiment, thedeprotection occurs at specified locations on the chip using lightshined through a reticle to interact with a photolabile compound, e.g.,a photobase or photoacid, which deprotects the protected amine group.

Examples

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1: AECL Wafer Processing

Steps 1-7 are described with reference to FIG. 10A; steps 8-14 aredescribed with reference to FIG. 10B.

Step 1: Silicon wafers were obtained from University wafers. 1000Asilicon dioxide was deposited using thermal oxide deposition in anoxidation chamber.

Step 2: P5107 (photoresist) obtained from Rohm and Haas were coated onthe wafers using a RF3 S Sokudo coater. Using a working electrode photomask, these wafers were exposed in a Nikon 5205 DUV at 18 mj/cm2. Thewafers were developed in a developer for 60 s. Oxide etch was performedusing Hydrofluoric acid (HF) bath for 30 s to remove 1000A oxide.Photoresist was stripped using Acetone wash followed by Isopropanol washfor 30 s each in a coater. All solvents and HF were obtained from SigmaAldrich.

Step 3: Uniform thickness of 1500A Gold was deposited on top of thiswafer substrate by sputtering.

Step 4: The wafers were polished in a chemical mechanical planarization(CMP) polisher until oxide layer was reached.

Step 5: P5107 photoresist obtained from Rohm and Haas were coated on thewafers using a RF3 S Sokudo coater. Using a counter electrode photomask, these wafers were exposed in a Nikon 5205 DUV at 18 mj/cm2. Thewafers were developed in a developer for 60 s. Oxide etch was performedusing Hydrofluoric acid (HF) bath to remove 1000A oxide. Photoresist wasstripped using Acetone wash followed by Isopropanol wash for 30 s eachin a coater.

Step 6: Uniform thickness of 1500A copper was deposited on top of thiswafer substrate by sputtering.

Step 7: The wafers were polished in a CMP polisher until oxide layer wasreached.

Step 8: Thermal oxide of 1000A was grown on top of the wafers in anoxidation chamber. P5107 photoresist obtained from Rohm and Haas werecoated on the wafers using a RF3 S Sokudo coater. Using a interconnectphoto mask, these wafers were exposed in a Nikon S205 DUV at 18 mj/cm2.The wafers were developed in a developer for 60 s. Oxide etch wasperformed using Hydrofluoric acid (HF) bath to remove 1000A oxide.Photoresist was stripped using Acetone wash followed by Isopropanolwash.

Step 9: Uniform thickness of 500A Aluminum interconnect was deposited ontop of this wafer substrate by sputtering.

Step 10: The wafers were polished in a CMP polisher until the electrodeslayer was reached.

Step 11: P5107 photoresist obtained from Rohm and Haas was coated on thewafers using a RF3 S Sokudo coater. Using an output photo mask, thesewafers were exposed in a Nikon S205 DUV at 18 mj/cm2. The wafers weredeveloped in a developer for 60 s. Oxide etch was performed usingHydrofluoric acid (HF) bath to remove 1000A oxide. Photoresist wasstripped using Acetone wash followed by Isopropanol wash.

Step 12: Uniform thickness of 500A copper was deposited on top of thiswafer substrate by sputtering.

Step 13: The wafers were polished in a CMP polisher until oxide layerwas reached.

Step 14: The wafer was then flipped and photoresist was coated on thewafer backside. Using a photo mask, these wafers were exposed in a NikonS205 DUV at 18 mj/cm2. The wafers were developed in a developer for 60s. Silicon etch was performed using Hydrofluoric acid (HF) bath toremove silicon until the contacts were reached. Photoresist was strippedusing Acetone wash followed by Isopropanol wash.

Example 2: Functionalization of Wafer and Dicing into Chips Production

Wafers comprising electrodes as shown in FIGS. 10A and B for supplyingvoltage to array features were provided according to Example 1. ACOOH-functionalized surface was formed as follows on an AECL wafer:

11-Mercaptoundecanoic acid and Acetic Acid were obtained from SigmaAldrich. Ethanol, Hydrogen Peroxide, Sulfuric Acid are obtained fromVWR.

To functionalize with COOH groups, the AECL wafers of Example 1, havinggold working electrodes, were cleaned with piranha solution whichcomprises 50 weight % of pure Sulfuric acid and 50 weight % of HydrogenPeroxide for 60 minutes. The wafers were then rinsed with DI Watercontinuously for 5 minutes followed by rinsing with Ethanol for 5minutes. The wafers were washed with a mixture of 50% Ethanol and 50% DIWater for 10 minutes. The wafers were then contacted with a solutioncontaining 2.5 weight % of 11-Mercaptoundecanoic acid and 97.5 weight %of Pure Ethanol for 12 hours under mild shaking conditions. After 10-12hours, wafers were then rinsed for Ethanol and isopropanol (IPA) for 5minutes each. This was followed by washing the wafers with DI Water for10 minutes and hot acetic acid solution which was prepared by mixing 10weight % of Acetic acid in 90 weight % of DI Water at 60 C for 45minutes. Finally, the wafers was rinsed with DI Water and IPA for 5minutes each and blown dry under nitrogen. Following this step, thewafers were diced into chips of 3.0 mm×3.0 mm.

Example 3: Chip Activation and Anti-TNF-Alpha Antibody Coupling

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC],N-hydroxysuccinimide [NHS], Ethanolamine and Phosphate Buffer Saline(PBS) buffer were obtained from Sigma Aldrich. Primary and secondaryanti TNF-alpha antibodies and TNF-alpha were obtained from ABCAM. Anactivation solution of EDC and NHS was prepared by dissolving 4% byweight of EDC and 2% by weight of NHS in deionized water. The activationsolution was then applied to the surface of the wafer at roomtemperature for 10 minutes. The chips were then washed with deionizedwater for 3 minutes.

The primary anti-TNF alpha antibody was coupled to the chip by adding asolution of 10 ug/ml of antibody in PBS buffer to the surface of thewafer with activated COOH groups for 30 mins, resulting in binding ofthe COOH groups to free amine of the primary antibody. This was followedby capping of unreacted carboxylic acid groups on the surface with 5weight % Ethanolamine in 95 weight % DI water for 10 minutes followed bywashing the wafer in DI water for 10 minutes.

Example 4: Prototype Pillar Mount for AECL Biochip

FIG. 11A shows steps for preparing a pillar mount for an AECL biochip.

Step 1: Silicon wafers were obtained from University wafers. 1000Asilicon dioxide was deposited using thermal oxide deposition in anoxidation chamber.

Step 2: P5107 (photoresist) obtained from Rohm and Haas were coated onthe wafers using a RF3 S Sokudo coater. Using an AECL pillar workingelectrode photo mask, these wafers were exposed in a Nikon S205 DUV at18 mj/cm2. The wafers were developed in a developer for 60 s. Oxide etchwas performed using Hydrofluoric acid (HF) bath for 30 s to remove 1000Aoxide. Photoresist was stripped using Acetone wash followed byIsopropanol wash for 30 s each in a coater. All solvents and HF wereobtained from Sigma Aldrich.Step 3: Uniform thickness of 1500A Gold was deposited on top of thiswafer substrate by sputtering. The wafers were polished in a chemicalmechanical planarization (CMP) polisher until oxide layer was reached.Step 4: P5107 photoresist obtained from Rohm and Haas were coated on thewafers using a RF3S Sokudo coater. Using an AECL pillar counterelectrode photo mask, these wafers were exposed in a Nikon S205 DUV at18 mj/cm2. The wafers were developed in a developer for 60 s. Oxide etchwas performed using Hydrofluoric acid (HF) bath to remove 1000A oxide.Photoresist was stripped using Acetone wash followed by Isopropanol washfor 30 s each in a coater.Step 5: Uniform thickness of 1500A copper was deposited on top of thiswafer substrate by sputtering. The wafers were polished in a CMPpolisher until oxide layer was reached.

FIG. 11B shows a top view of a pillar mount used for an AECL biochip,prepared according to the steps outlined above in this example. To testthe performance of the AECL-TNF alpha chips, an AECL biochip was mountedon the pillar as follows:

The AECL biochip's working and counter electrodes were picked and placedover the AECL pillar mount. Positional correspondence and electricalcontact between the pillar mount and chip working and counter electrodeswere stabilized using conductive tape obtained from 3M. The AECL pillarmount working and counter electrodes were connected via copper clips toa model XP-100 voltage controller from Elenco which supplies from 1.5 to12V.

Example 5: Preparation of an AECL Tag

This example describes preparation of an amplifiedelectrochemiluminescent tag. In this example, ruthenium bis(2,2bipyridine) bis (2,2 dicarboxylic acid ester) is theelectrochemiluminescent moiety, and tyramide is the signal amplificationmoiety (FIG. 1) through which a plurality of AECL tags bind to targetmolecules in the vicinity of peroxidase activity (e.g., HRP enzyme) andan oxidizing agent (e.g., hydrogen peroxide).

50 ul of 0.01M of tyramine.HCL and 50 ul of 0.01M of Ruthenium bis(2,2bipyridine) bis (2,2 dicarboxylic acid) ester were mixed in DI waterwith the presence of 5 ul of N,N-Diisopropylethylamine (DIEA). Themixture was shaken on a rotary mixer set at 400 rpm for 2 hours,followed by addition of 1 ul of ethanolamine and then shaken again foran additional 10 minutes. TLC was used to purify the solution and theresulting solution was desalted and lyophilized to obtain 0.56 mg of theAECL tag shown in FIG. 1. The AECL tag was dissolved in volume of PBSbuffer to generate a 0.5 mg/mL stock solution.

Example 6: TNF-Alpha AECL Assay

TNF alpha was dissolved in varying concentration of 1 fg/mL to 100 ng/mLin PBST (PBST contains 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mMNaCl, 0.05% Tween® 20, pH 7.4) and was added to the AECL chips on thepillar substrate. This was incubated for 30 mins at 37c. After this, thechips were washed with PBST buffer for 5 mins. A secondary TNF-alphaAb-HRP conjugate obtained from ABcam, was added in a dilution of 1:1000in PBST and incubated for 15 mins at 37c. Then a tag solution was madethat included a 1:10 dilution in PBS of the AECL tag stock solution and0.003 weight % hydrogen peroxide. This was added to the chips resultingin binding of multiple AECL tags to the captured TNF alpha/antibody-HRPcomplexes. Tripropylamine (TPA) was added to the chip in a concentrationof 0.1M in an 0.02% sodium acetate buffer with Tween 20. The electricalpotential at working electrode was ramped from 0 to 3.5V. The intensityof AECL was read by a CCD camera at 620 nm. A similar assay was alsoconducted that differed by using an ECL tag obtained from Meso ScaleDiagnostics. The data from both assays are shown in FIG. 12. The AECLtag can clearly detect TNF alpha in sub-picogram/mL ranges whereas anECL tag can only detect pictogram/mL level ranges.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1.-28. (canceled)
 29. An AECL tag composition, comprising:


30. The AECL tag composition of claim 29, wherein M^(n+) is Ru²⁺.
 31. Asolid state microarray for detecting target biomolecules, comprising: anaddressable network of working electrodes and counter electrodesintegral to said microarray, wherein said working electrodes comprisechemically-functionalized surfaces comprising functional groups, saidfunctional groups capable of covalently binding capture ligands atdefined locations on a substrate, and said capture ligands capable ofspecifically binding said target biomolecules thereby immobilizing saidtarget biomolecules at said defined locations on said substrate.
 32. Thesolid state microarray of claim 31, wherein saidchemically-functionalized surfaces comprise COOH or NH2 functionalgroups.
 33. (canceled)
 34. The solid state microarray of claim 31,wherein said functional groups are covalently bound to capture ligands.35. The solid state microarray of claim 34, wherein said capture ligandsare selected from the group consisting of peptides and proteins.
 36. Thesolid state microarray of claim 35, wherein said proteins are antigenbinding proteins or antibodies.
 37. (canceled)
 38. The solid statemicroarray of claim 31, further comprising a plurality of biomolecularcomplexes bound to said working electrodes, said biomolecular complexescomprising at least 2, 5, 10, 20, 50, 100, 200, 500, or 1,000 bound AECLtags.
 39. The solid state microarray of claim 38, wherein said AECL tagcomprises a metal chelate.
 40. (canceled)
 41. The solid state microarrayof claim 39, wherein said metal chelate is ruthenium.
 42. The solidstate microarray of claim 38, wherein said AECL tag comprises tris(bipyridine)ruthenium(II).
 43. The solid state microarray of claim 39,wherein said AECL tag further comprises tyramide bound to said metalchelate.
 44. The solid state microarray of claim 39, wherein said AECLtag is


45. The solid state microarray of claim 38, wherein an electricalpotential difference exists between at least one pair of working andcounter electrodes said potential difference generatingelectrochemiluminescence from said AECL tags.
 46. The solid statemicroarray of claim 31, wherein the array comprises at least 2, 4, 8,16, 32, 64, 100, 200, 500, 1000, 2000, 5000, 10,000, 15,000, 20,000,30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 1,000,000,1,500,000, 2,000,000 or more working electrodes.
 47. An assay plate formounting a solid state microarray, said assay plate comprising: apillar, said pillar comprising a top surface and a bottom surface, saidtop surface comprising a mounting surface to receive a solid statemicroarray of claim 31 and at least one working electrode and onecounter electrode configured to contact and to be in electricalcommunication with a corresponding at least one working electrode andcounter electrode on a bottom surface of said solid state microarray,said bottom surface of said pillar comprising contacts for supplyingpower to said at least one pillar working electrode and counterelectrode.
 48. The assay plate of claim 47, comprising a plurality ofpillars.
 49. The assay plate of claim 48, wherein said plurality is 24,96, 384 or
 1586. 50. The assay plate of claim 47, wherein each of saidpillars comprises at least 2, 4, 8, 16, 32, 64, 100, 200, 500, 1000,2000, 5000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000, 1,000,000, 1,500,000, 2,000,000 or more workingelectrodes.
 51. An assembly, comprising a solid state microarray thatcomprises: an addressable network of working electrodes and counterelectrodes integral to said microarray, said working electrodes comprisechemically-functionalized surfaces comprising functional groups, whereinsaid solid state microarray is mounted on said pillar mounting surfaceof said assay plates of claim 47, and wherein said working electrodesand said counter electrodes of said solid state microarrays are inelectrical communication with said working electrodes and said counterelectrodes of said pillars.
 52. The assembly of claim 51, wherein atleast 50, 100, 200, or 500 separate analytes can be detected on one ormore solid state microarrays mounted onto a single pillar.
 53. The assayplate or assembly of claim 52, further comprising an assay capconfigured to engage with said assay plate and comprising pillar strutsmounted to pillar walls, wherein said pillar struts are configured toslide into corresponding grooves on the sides of said assay platepillars and thereby position said pillar walls on top of said pillars.54. The assay plate or assembly of claim 53, wherein said pillar wallscomprise a reservoir for containing an assay solution in contact withsaid solid state microarray.